Complex of cell translocational peptide and magnetic nanoparticles and use thereof

Abstract

The present invention relates to a cell-penetrating peptide/fluorescence-labeled magnetic nanoparticle complex and the use thereof. More specifically, relates to a cell-penetrating peptide/fluorescence-labeled magnetic nanoparticle complex in which a cell-penetrating peptide is chemically linked to a fluorescence-labeled magnetic nanoparticle, such that fluorescence-labeled magnetic nanoparticle can be stably introduced directly into cells without endocytosis, and a composition for cell imaging containing the complex. The disclosed invention suggests an innovative therapeutic technology which shows high stability, maximizes the effect of imaging diagnosis through optimal targeting and minimizes side effects, unlike existing viral peptide transporters.

1 . A method for preparing a complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle, the method comprising the steps of:
 (a) preparing an amino group-containing magnetic nanoparticle by modifying the surface of magnetic nanoparticle with an amino group;   (b) preparing a fluorescence-labeled magnetic nanoparticle by adding a fluorescent substance to the amino group-containing magnetic nanoparticle; and   (c) preparing cell-penetrating peptide/fluorescence-labeled magnetic nanoparticle complex by linking a cell-penetrating peptide to the fluorescence-labeled magnetic nanoparticle.   
     
     
         2 . The method for preparing a complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle according to  claim 1 , wherein the cell-penetrating peptide is a peptide in which the content of an amino acid selected from the group consisting of arginine, lysine and histidine is 70-80%. 
     
     
         3 . The method for preparing a complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle according to  claim 1 , wherein the cell-penetrating peptide is selected from the group consisting of low molecular weight protamine (LMWP, SEQ ID NO: 1: VSRRRRRRGGRRRR), TAT (SEQ ID NO: 2: YGRKKRRQRRR), penetratin (SEQ ID NO: 3: RQIKIWFQNRRMKWKK), antennapedia (ANTP), D-arginine oligopeptide (R 8  or more) and L-arginine oligopeptide (R 8  or more). 
     
     
         4 . The method for preparing a complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle according to  claim 1 , wherein the magnetic nanoparticle is selected from the group consisting of ferric ammonium citrate (FAC), ferrum oxide, superparamagnetic iron oxide nanoparticle (SPIO), carboxy-dextran coated iron oxide nanoparticle, ultrasmall superparamagnetic iron oxide nanoparticle (USPIO), monocrystalline iron oxide nanoparticles (MION), polycrystalline iron oxide nanoparticles (PION) and oral magnetic particles (OMP). 
     
     
         5 . The method for preparing a complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle according to  claim 1 , wherein the diameter of said magnetic nanoparticle is 10˜150 nm. 
     
     
         6 . The method for preparing a complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle according to  claim 1 , wherein said linking of step (c) is performed by a crosslinker. 
     
     
         7 . The method for preparing a complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle according to  claim 6 , wherein the crosslinker is selected from the group consisting of 1,4-bis-maleimidobutane (BMB), 1,11-bis-maleimidotetraethyleneglycol (BM[PEO]4), 1-ethyl-3-[3-dimethyl aminopropyl]carbodiimide hydrochloride (EDC), succinimidyl-4-[N-maleimidomethylcyclohexane-1-carboxy-[6-amidocaproate]] (SMCC) and sulfonate thereof (sulfo-SMCC), succimidyl 6-[3-(2-pyridyldithio)-ropionamido]hexanoate (SPDP) and sulfonate thereof (sulfo-SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and sulfonate thereof (sulfo-MBS), succimidyl[4-(p-maleimidophenyl)butyrate] (SMPB) and sulfonate (sulfo-SMPB) thereof. 
     
     
         8 . The method for preparing a complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle according to  claim 1 , wherein the fluorescent substance is selected from the group consisting of fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate (TRITC), alexa fluor, rhodamine red-X, texas red, tetramethylrhodamine, cascade blue, DAPI(4′,6-diamidino-2-phenylindole), coumarine, lucifer yellow, and dansylaminde. 
     
     
         9 . The method for preparing a complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle according to  claim 1 , wherein said modifying the surface of the magnetic nanoparticle of step (a) is carried out using one selected from the group consisting of ammonium hydroxide, ethanol, aminopropyl trimethylsilane (APTMS), N-(3-dimethylpropyl)-N-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS) and N-hydroxysuccinimide (NHS). 
     
     
         10 . The method for preparing a complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle according to  claim 1 , which further comprising the step of adjusting pH to neutral by adding sodium citrate to the amino group-containing magnetic nanoparticle prepared in step (a). 
     
     
         11 . A complex of a cell-penetrating peptide and a fluorescence-labeled magnetic nanoparticle, which is prepared by the method of  claim 1 , and in which the cell-penetrating peptide is linked to the fluorescence-labeled magnetic nanoparticle, wherein the diameter of said complex is 10-50 nm. 
     
     
         12 . A composition for cell imaging containing the complex in which the cell-penetrating peptide is linked to the fluorescence-labeled magnetic nanoparticle of  claim 11 .